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Specific antibody responses against membrane proteins of erythrocytes infected by Plasmodium falciparum of individuals briefly exposed to malaria.

Fontaine A, Pophillat M, Bourdon S, Villard C, Belghazi M, Fourquet P, Durand C, Lefranc D, Rogier C, Fusai T, Almeras L - Malar. J. (2010)

Bottom Line: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group.Some of these antigenic proteins were identified by an original immuno-proteomic approach.Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de Recherche en Biologie et Epidémiologie Parasitaires (URBEP)-UMR6236-IFR48, Antenne Marseille de l'Institut de Recherche Biomédicale des Armées (IRBA), BP 60109, 13262 Marseille Cedex 07, France.

ABSTRACT

Background: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers.

Methods: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission.

Results: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach.

Conclusion: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

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Related in: MedlinePlus

Schematic representation of experimental workflow for an accurate identification of antigenic protein using 2-D immunoblot with a fluorescence-based method. The different steps are numbered from #1 to #6. Briefly, iRBC membrane protein extracts were pre-labelled with Cy5 cyanine (#1), separated by 2-D electrophoresis (#2), electroblotted onto membranes, probed with the pool from 5 selected BEI sera and incubated with a goat-anti-human IgG FITC-conjugate (#3). Following blot digitalization at Cy5 and FITC wavelengths (#4), two images corresponding respectively to the antigenic protein pattern and the total protein expression profile were obtained. The superimposition of these two images (#5) allowed us to excize accurately spots of interest on preparative gel run in parallel (#3'), before to submit them to mass spectrometry (MS) for identification (#6). Protein expression profile was used as "internal standard" to perform a perfect match between the blot and the preparative gel.
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Figure 3: Schematic representation of experimental workflow for an accurate identification of antigenic protein using 2-D immunoblot with a fluorescence-based method. The different steps are numbered from #1 to #6. Briefly, iRBC membrane protein extracts were pre-labelled with Cy5 cyanine (#1), separated by 2-D electrophoresis (#2), electroblotted onto membranes, probed with the pool from 5 selected BEI sera and incubated with a goat-anti-human IgG FITC-conjugate (#3). Following blot digitalization at Cy5 and FITC wavelengths (#4), two images corresponding respectively to the antigenic protein pattern and the total protein expression profile were obtained. The superimposition of these two images (#5) allowed us to excize accurately spots of interest on preparative gel run in parallel (#3'), before to submit them to mass spectrometry (MS) for identification (#6). Protein expression profile was used as "internal standard" to perform a perfect match between the blot and the preparative gel.

Mentions: To further characterize these discriminatory antigens, a 2-D immunoproteomic approach was adopted. A major difficulty of this approach is to perform a perfect match between antigenic spots detected on 2-D immunoblot and their counterparts on the preparative gel, on which matched spots will be excized and further analysed by mass spectrometry (MS). As the results of these protein identifications are the starting point of further studies, it is necessary to limit miss matching. Thus, 2-D immunoblot using a fluorescence-based method allows to increase considerably the confidence in spot matching between preparative gel and immunoblots (Figure 3).


Specific antibody responses against membrane proteins of erythrocytes infected by Plasmodium falciparum of individuals briefly exposed to malaria.

Fontaine A, Pophillat M, Bourdon S, Villard C, Belghazi M, Fourquet P, Durand C, Lefranc D, Rogier C, Fusai T, Almeras L - Malar. J. (2010)

Schematic representation of experimental workflow for an accurate identification of antigenic protein using 2-D immunoblot with a fluorescence-based method. The different steps are numbered from #1 to #6. Briefly, iRBC membrane protein extracts were pre-labelled with Cy5 cyanine (#1), separated by 2-D electrophoresis (#2), electroblotted onto membranes, probed with the pool from 5 selected BEI sera and incubated with a goat-anti-human IgG FITC-conjugate (#3). Following blot digitalization at Cy5 and FITC wavelengths (#4), two images corresponding respectively to the antigenic protein pattern and the total protein expression profile were obtained. The superimposition of these two images (#5) allowed us to excize accurately spots of interest on preparative gel run in parallel (#3'), before to submit them to mass spectrometry (MS) for identification (#6). Protein expression profile was used as "internal standard" to perform a perfect match between the blot and the preparative gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959075&req=5

Figure 3: Schematic representation of experimental workflow for an accurate identification of antigenic protein using 2-D immunoblot with a fluorescence-based method. The different steps are numbered from #1 to #6. Briefly, iRBC membrane protein extracts were pre-labelled with Cy5 cyanine (#1), separated by 2-D electrophoresis (#2), electroblotted onto membranes, probed with the pool from 5 selected BEI sera and incubated with a goat-anti-human IgG FITC-conjugate (#3). Following blot digitalization at Cy5 and FITC wavelengths (#4), two images corresponding respectively to the antigenic protein pattern and the total protein expression profile were obtained. The superimposition of these two images (#5) allowed us to excize accurately spots of interest on preparative gel run in parallel (#3'), before to submit them to mass spectrometry (MS) for identification (#6). Protein expression profile was used as "internal standard" to perform a perfect match between the blot and the preparative gel.
Mentions: To further characterize these discriminatory antigens, a 2-D immunoproteomic approach was adopted. A major difficulty of this approach is to perform a perfect match between antigenic spots detected on 2-D immunoblot and their counterparts on the preparative gel, on which matched spots will be excized and further analysed by mass spectrometry (MS). As the results of these protein identifications are the starting point of further studies, it is necessary to limit miss matching. Thus, 2-D immunoblot using a fluorescence-based method allows to increase considerably the confidence in spot matching between preparative gel and immunoblots (Figure 3).

Bottom Line: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group.Some of these antigenic proteins were identified by an original immuno-proteomic approach.Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de Recherche en Biologie et Epidémiologie Parasitaires (URBEP)-UMR6236-IFR48, Antenne Marseille de l'Institut de Recherche Biomédicale des Armées (IRBA), BP 60109, 13262 Marseille Cedex 07, France.

ABSTRACT

Background: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers.

Methods: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission.

Results: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach.

Conclusion: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

Show MeSH
Related in: MedlinePlus